MODULE2

 

 

Authors : Patrice Dosset, Martin Blackledge

 

Institut de Biologie Structurale, Grenoble, France

 

Developed in the context of EU NMR Joint Research Project JRA3 ORIENTING NMR

 

 

Analysis of residual anisotropic interactions measured in proteins and nucleic acids aligned in dilute liquid crystalline media.

 

Version 2

–      Allows for the analysis of residual chemical shifts due to non-averaged chemical shift anisotropy.

–      Allows for the analysis and comparison of multiple data sets measured for the same molecule.

–      Allow for output of fitted data into external files (bug-fix).

–      Allows for cursor selection of RDC/RCS between peptide units aswell as within peptide units.

 

For residual dipolar coupling based analysis see main manual in download package.

 

The executable is currently for Linux machines. A Macintosh executable will be available shortly.

 


Data format :

 

 

3      C       4       N                 -5.5975         1.0000

4      C       5       N                 -56.3931                1.0000

5      C       6       N                 -88.1987                1.0000

6      C       7       N                 -35.2552                1.0000

10     C       11      N                 -0.0012         1.0000

11     C       12      N                 12.2747         1.0000

12     C       13      N                 34.8201         1.0000

13     C       14      N                 62.4405         1.0000

14     C       15      N                 -56.8725                1.0000

 

Two atoms identify the nucleus for which the CSA is to be taken into account.

The first atom is the appropriate nucleus. The second defines the neighbour with respect to which the angle q describing the orientation of the tensor eigenvalues in the plane. In the case shown the CSA is for the Carbonyl carbon and the orientation of the eigenvalues of the CSA tensor is defined by the angle q, defined by a clockwise rotation about the norm with respect to the plane, relative to the vector C-N (see figure).

The experimental value is shown in ppb. The uncertainty is in the final column.

 

1) Fitting of residual anisotropic interactions : Either Chemical shifts, dipolar couplings or both can be included in the fit

 

 

 

2) Fitting of residual anisotropic interactions : Either alignment tensor or average chemical shift tensors can be fit to the data.

 

 

 

The eigenvalues and eigenvectors can be either set by the use or fit. The precision of the parameter determination can be estimated using Monte Carlo simulations.

 

3) Standard RDC analysis – as in MODULE Vsn 1 (see manual for version 1).

RCSA analysis is presented in a very similar way :

 

4) Once the data have been read, they are identified above, or between the appropriate parts of the primary sequence using the Visualisation menu as points Ô.Õ for RDCs and circumflex Ô^Õ for RCSA data. They can be individually selected using the cursor and the data fitting menus. The molecule can of course be dissected into different domains to analyze the fitting these domains and to reorient with respect to a common alignment frame.

 

 

 

 

 

5) Fitting of the CSA tensors. It is not possible to fit the alignment and CSA tensors to the same data when there are no RDC data available (in this case the solution is not defined). But in the case where RDC data are available you can fit both tensors. Individual components of the tensor (either eigenvectors or the orientation of the eigenvectors in the peptide plane), or all three parmeters, can be fitted. The results of the fit can be examined in the text window from which the program was run. The quality of the fitting can be examined in the Visualisation Correlation menu, as for RDC analysis. Of course it is possible to determine the optimal CSA tensor for different domains of the molecule, for example for alpha helices or for beta sheets as in the example below :

 

 

 

 

 

 

 

Some references to the use of residual CSA measurement and analysis in proteins and nucleic acids (a non-exhaustive list) :

 

W.Y. Choy, M. Tollinger, G.A. Mueller, et al. Direct structure refinement of high molecular weight proteins against residual dipolar couplings and carbonyl chemical shift changes upon alignment: an application to maltose binding protein. J. Biomol. NMR 21, (2001) 31-40

 

N. Lancelot, K. Elbayed, M Piotto. Applications of variable-angle sample spinning experiments to the measurement of scaled residual dipolar couplings and N-15 CSA in soluble proteins J. Biomol. NMR 33, (2005) 153-161

 

G. Cornilescu, A. Bax, Measurement of proton, nitrogen, and carbonyl chemical shielding anisotropies in a protein dissolved in a dilute liquid crystalline, J. Am. Chem. Soc. 122 (2000),  10143–10154.

 

J. Boyd, C. Redfield, Characterization of 15N chemical shift anisotropy from orientation-dependent changes to 15N chemical shifts in dilute bicelle solutions, J. Am. Chem. Soc. 121 (1999),  7441–7442.

 

G. Cornilescu, J. Marquardt, M. Ottiger, A. Bax, Validation of protein structure from anisotropic carbonyl chemical shifts in a dilute liquid crystalline phase, J. Am. Chem. Soc. 120 (1998),  6836–6837.

 

J. Kurita, H. Shimahara, N. Utsunomiya-Tate, S. Tate, Measurement of 15N chemical shift anisotropy in a protein dissolved in a dilute liquid crystalline medium with the application of magic angle sample spinning, J. Magn. Reson. 163 (2003),  163–173.

 

A. Grishaev, JF Ying, A. Bax. Pseudo-CSA restraints for NMR refinement of nucleic acid structure. J. Am. Chem. Soc. 128, 31, (2006) 10010-10011.

 

G. Bouvignies, S. Meier, S. Grzesiek and M. Blackledge. Ultra-High Resolution Backbone Structure of Perdeuterated Protein GB1 using Residual Dipolar Couplings from Two Alignment Media. Angewandte Chemie International Edition. 45, (2006) 8166-8169.